High-pressure study of magnetic nanoparticles with a polyelectrolyte brush as carrier particles for enzymes.

The recovery of enzymes from a reaction medium can be achieved in a convenient way by using magnetic nanoparticles (MNP) as carriers. Here, we present MNP with a polyelectrolyte brush composed of poly(ethylene imine) (PEI) to provide a benign environment for the immobilized enzyme molecules. Yeast alcohol dehydrogenase (ADH) has been tested for enzymatic activity when it is free in solution or adsorbed on the PEI brush-MNP. Furthermore, the effect of pressure on the enzymatic activity has been studied to reveal activation volumes, which are a sensitive probe of the transition state geometry. The results of this study indicate that the secondary structure of ADH is pressure-stable up to 9 kbar. The enzymatic activity of ADH can be analyzed using Michaelis-Menten kinetics free in solution and adsorbed on the PEI brush-MNP. Remarkably, no significant changes of the Michaelis constant and the activation volume are observed upon adsorption. Thus, it can be assumed that the turnover number of ADH is also the same in the free and adsorbed state. However, the maximum enzymatic rate is reduced when ADH is adsorbed, which must be explained by a lower effective enzyme concentration due to steric hindrance of the enzyme inside the PEI brush of the MNP. In this way, the pressure experiments carried out in this study enable a distinction between steric and kinetic effects on the enzymatic rate of adsorbed ADH.

Analyzing protein-ligand and protein-interface interactions using high pressure.

All protein function is based on interactions with the environment. Proteins can bind molecules for their transport, their catalytic conversion, or for signal transduction. They can bind to each other, and they adsorb at interfaces, such as lipid membranes or material surfaces. An experimental characterization is needed to understand the underlying mechanisms, but also to make use of proteins in biotechnology or biomedicine. When protein interactions are studied under high pressure, volume changes are revealed that directly describe spatial contributions to these interactions. Moreover, the strength of protein interactions with ligands or interfaces can be tuned in a smooth way by pressure modulation, which can be utilized in the design of drugs and bio-responsive interfaces. In this short review, selected studies of protein-ligand and protein-interface interactions are presented that were carried out under high pressure. Furthermore, a perspective on bio-responsive interfaces is given where protein-ligand binding is applied to create functional interfacial structures.

Interaction of calmodulin with poly(acrylic acid) brushes: Effects of high pressure, pH-value and ligand binding.

Poly(acrylic acid) (PAA) brushes are well-known to interact with proteins in an ionic strength-dependent way. Moreover, they provide a native-like environment that largely maintains the secondary structure and biological activity of adsorbed proteins. Recently, it has been shown that the application of high pressure can lead to a reduced protein adsorption at a PAA brush in the case of a positively charged protein. Here, we analyze the effect of pressure on the interactions between a protein and a PAA brush in more detail. We use calmodulin as model protein that has a negative net charge at neutral pH-value and determine the degree of adsorption at a planar PAA brush applying total internal reflection fluorescence (TIRF) spectroscopy. Remarkably, the degree of calmodulin adsorption at a PAA brush is increasing with increasing pressure, when the protein is negatively charged. However, at low pH-value, where calmodulin is positively charged, high pressure leads to a partial desorption of the protein. Moreover, in the presence of trifluoperazine, which binds to calmodulin as a ligand, the pressure effect is diminished. The results of this study indicate that protein adsorption at a PAA brush at the "wrong" side of the isoelectric point, i.e. under net electrostatic repulsion, can involve a volume reduction that is favored under high pressure. It is suggested that this volume reduction is related to a hydration of counterions that are released from the PAA chains and the protein surface. In contrast, at pH-values close to the isoelectric point, the obtained data are consistent with a charge regulation mechanism that involves a volume increase. Thus, the application of high pressure in combination with pH-variation, as carried out in this study, provides the volume changes of adsorption that need to be consistent with any proposed mechanism of protein interaction with a PAA brush.

Lipid Phase Control and Secondary Structure of Viral Fusion Peptides Anchored in Monoolein Membranes.

The fusion of lipid membranes involves major changes of the membrane curvatures and is mediated by fusion proteins that bind to the lipid membranes. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different viral fusion peptides, HA2-FP and TBEV-FP, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). By using synchrotron X-ray diffraction, the changes of the monoolein phase behavior upon binding the peptides have been determined and the concomitant secondary structures of the peptides have been analyzed by FTIR spectroscopy. As main results we have found that the fusion peptides interact differently with monoolein and change the pressure and temperature dependent lipid phase behavior to different extents. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. These peptide-induced phase changes can partially be reversed by the application of high pressure, demonstrating that the promotion of negative curvature is achieved by a less dense packing of the monoolein membranes by the fusion peptides. Pressure jumps across the cubic-lamellar phase transition reveal that HA2-FP has a negligible effect on the rates of the cubic and the lamellar phase formation. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid-fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

Volume profile of α-chymotrypsin during adsorption and enzymatic reaction on a poly(acrylic acid) brush.

Poly(acrylic acid) (PAA) brushes are known to provide a native-like environment for proteins. In this study, we explore this biocompatibility under high pressure conditions. Using α-chymotrypsin (α-CT) as a model enzyme, we report on the pressure dependencies of the enzymatic activity and the neutron scattering length density profile, when this enzyme is adsorbed on a PAA brush. From high pressure total internal reflection fluorescence spectroscopy, an increasing enzymatic activity has been observed up to 1000 bar, but a rather pressure independent enzymatic activity at higher pressures up to 2000 bar. This finding suggests a non-constant activation volume of α-CT on the PAA brush that is negative below 1000 bar. Thus, the compact nature of the transition state of α-CT is largely preserved upon adsorption. We have also performed high pressure neutron reflectivity experiments to determine the spatial distribution of α-CT inside the PAA brush. Apparently, the enzyme is strongly binding to the PAA chains with 2.3 mg m(-2) of adsorbed enzyme that is reduced to about 1.7 mg m(-2) at 1000-2000 bar. This change of adsorbed mass is consistent with a positive volume change of adsorption, which is probably reflecting electrostriction upon protein-PAA interaction. Thus, the performed high pressure experiments provide new insights into the volume profile of α-CT during adsorption and enzymatic activity on the PAA brush. They also demonstrate that the biocompatible properties of a PAA brush can even be enhanced by pressure.